Excessive leaf oil modulates the plant abiotic stress response via reduced stomatal aperture in tobacco ( <i>Nicotiana tabacum</i> )

High lipid producing (HLP) tobacco (Nicotiana tabacum) is a potential biofuel crop that produces an excess of 30% dry weight as lipid bodies in the form of triacylglycerol. While using HLP tobacco as a sustainable fuel source is promising, it has not yet been tested for its tolerance to warmer environments that are expected in the near future as a result of climate change. We found that HLP tobacco had reduced stomatal conductance, which results in increased leaf temperatures up to 1.5°C higher under control and high temperature (38°C day/28°C night) conditions, reduced transpiration, and reduced CO₂ assimilation. We hypothesize this reduction in stomatal conductance is due to the presence of excessive, large lipid droplets in HLP guard cells imaged using confocal microscopy. High temperatures also significantly reduced total fatty acid levels by 55% in HLP plants; thus, additional engineering may be needed to maintain high titers of leaf oil under future climate conditions. High-throughput image analysis techniques using open-source image analysis platform PlantCV for thermal image analysis (plant temperature), stomata microscopy image analysis (stomatal conductance), and fluorescence image analysis (photosynthetic efficiency) were developed and applied in this study. A corresponding set of PlantCV tutorials are provided to enable similar studies focused on phenotyping future crops under adverse conditions.